Methods and systems for dna isolation on a microfluidic device

ABSTRACT

The present invention relates to methods and systems for the isolation of DNA on a microfluidic device and the subsequent analysis of the DNA on the microfluidic device. More specifically, embodiments of the present invention relate to methods and systems for the isolation of DNA from patient samples on a microfluidic device and use of the DNA for performing amplification reactions, such as PCR, and detection, such as thermal melt analysis, on the microfluidic

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional of and claims priority to U.S. patentapplication Ser. No. 12/505,195, filed on Jul. 17, 2009, which isincorporated herein by reference in its entirety. This application isrelated to U.S. patent application Ser. No. 12/505,202, filed on Jul.17, 2009, entitled “METHODS AND SYSTEMS FOR MICROFLUIDIC DNA SAMPLEPREPARATION,” naming Weidong Cao, Hiroshi Inoue and Kevin Louder asinventors, which is incorporated herein by this reference in itsentirety.

BACKGROUND

1. Field of the Invention

The present invention relates to methods and systems for the isolationof DNA on a microfluidic device and the subsequent analysis of the DNAon the microfluidic device. More specifically, embodiments of thepresent invention relate to methods and systems for the isolation of DNAfrom patient samples on a microfluidic device and use of the DNA forperforming amplification reactions, such as PCR, and detection, such asthermal melt analysis, on the microfluidic device.

2. Description of Related Art

The detection of nucleic acids is central to medicine, forensic science,industrial processing, crop and animal breeding, and many other fields.The ability to detect disease conditions (e.g., cancer), infectiousorganisms (e.g., HIV), genetic lineage, genetic markers, and the like,is ubiquitous technology for disease diagnosis and prognosis, markerassisted selection, correct identification of crime scene features, theability to propagate industrial organisms and many other techniques.Determination of the integrity of a nucleic acid of interest can berelevant to the pathology of an infection or cancer. One of the mostpowerful and basic technologies to detect small quantities of nucleicacids is to replicate some or all of a nucleic acid sequence many times,and then analyze the amplification products. PCR is perhaps the mostwell-known of a number of different amplification techniques.

The basic steps of nucleic acid, such as DNA, isolation are disruptionof the cellular structure to create a lysate, separation of the solublenucleic acid from cell debris and other insoluble material, andpurification of the DNA of interest from soluble proteins and othernucleic acids. Historically, organic extraction (e.g.,phenol:chloroform) followed by ethanol precipitation was performed toisolate DNA. Disruption of most cells is performed by chaotropic salts,detergents or alkaline denaturation, and the resulting lysate is clearedby centrifugation, filtration or magnetic clearing. The DNA can then bepurified from the soluble portion of the lysate. When silica matricesare used, the DNA is eluted in an aqueous buffer such as Tris-EDTA (TE)or nuclease-free water.

DNA isolation systems for genomic, plasmid and PCR product purificationare historically based on purification by silica. Regardless of themethod used to create a cleared lysate, the DNA of interest can beisolated by virtue of its ability to bind silica in the presence of highconcentrations of chaotropic salts (Chen and Thomas, Anal Biochem101:339-341, 1980; Marko et al., Anal Biochem 121:382-387, 1982; Boom etal., J Clin Microbiol 28:495-503, 1990). These salts are then removedwith an alcohol-based wash and the DNA eluted in a low ionic strengthsolution, such as TE buffer or water. The binding of DNA to silica maybe driven by dehydration and hydrogen bond formation, which competesagainst weak electrostatic repulsion (Melzak et al., J Colloid andInterface Science 181:635-644, 1996). Hence, a high concentration ofsalt will help drive DNA adsorption onto silica, and a low concentrationwill release the DNA.

Recently, new methods for DNA purification have been developed whichtake advantage of the negatively charged backbone of DNA to a positivelycharged solid substrate (under specific pH conditions), and eluting theDNA using a change in solvent pH (ChargeSwitch® technology, Invitrogen,Corp., Carlsbad, Calif.; see, for example, U.S. Pat. No. 6,914,137 andInternational Published Application No. 2006/004611). Whatman has analternate technology (FTA® paper) that utilizes a cellulose based solidsubstrate impregnated with a lysis material that lyses cells,inactivates proteins, but captures DNA in the cellulose fibers, where itis retained for use in downstream applications (see, for example, U.S.Pat. No. 6,322,983).

The use of nuclear extracts was reported in 1983 (Dignani et al., NuclAcids Res 11:1475-1489, 1983). There are several commercial kits thatallow for the selective lysis of a cellular membrane while allowing forthe purification of cellular organelles, including nuclei. Sigma andPierce are two providers of commercial kits. These kits utilizecentrifugation for the collection of nuclei. There are two patents thatdescribe purifying nuclei from cells. U.S. Pat. No. 5,447,864 describesusing a DNA mesh to capture intact cell nuclei on a membrane to capturenuclei for various applications. A membrane is extended across theforward end of a pipette tip device and the DNA from lysed nuclei isused to capture the remaining intact nuclei. U.S. Pat. No. 6,992,181 B2describes the use of a CD device for the purification of DNA or cellnuclei. This method requires moving parts and centrifugal force toisolate DNA and or cell nuclei, using a barrier in the channel to impedeflow of DNA and nuclei. Both patents describe the capture of nucleiand/or DNA and subsequent steps of washing are also required for theirsystems.

Various papers have described the capture of nuclei or white blood cellsfor downstream use in PCR reactions. If only the white blood cells areisolated, the primary inhibitor of PCR reactions, haemoglobin, isremoved, yielding higher efficiencies in PCR, (Cheng et al., Nucl AcidsRes 24:380-385, 1996). Another approach (Service, Science 282:399-401,1998) involves mixing blood with a salt solution that lyses the cells.The lysate is then introduced into a chamber containing a glass wall onwhich DNA binds by charge interaction, while the rest of the sample isejected. DNA must then be washed and is eluted to another chamber.Another paper describes a microfluidic platform for cell separation andnucleus collection that uses dielectrophoresis (DEP) forces to separatecells in a continuous flow system. After a specific cell was captured alysis buffer was added and the nucleus of the cell can then be collectedto study nuclear proteins. The ability to perform PCR on a singlecaptured nucleus was demonstrated by Li et al. (Eukaryotic Cell2:1091-1098, 2003). A nucleus extracted by a micropipette was addeddirectly to a PCR reaction for the detection of a specific genesequence. This paper demonstrates that nuclei added directly to a PCRreaction can be used to deliver DNA template, and assays for specificgene targets can be conducted using nuclei isolated from cells.

The most significant problems with the current technologies are thatthey require specific buffers for DNA binding and washing, most of whichare not compatible with down stream applications such as PCR, and theyhave a wide range of efficiencies in the overall quantity of DNA that ispurified. This can be a significant problem when samples are to be usedin microfluidics. The multiple reagents that are typically required forDNA purification would demand that moving parts, such as valves, beconstructed into a microfluidic device for the introduction of multiplereagents in a solid phase extraction. In a microfluidic system, solidphase extraction or the use of multiple reagents is complicated and canlead to system failures. Commercial assays that are sold by Sigma andPierce for cellular organelle purification do not use a filtrationprocess for nuclei capture. Instead, they use centrifugal force tocollect the nuclei.

In addition, microfluidic devices have been designed to do cell sortingof whole blood and separating white blood cells from red blood cells andplasma. However, such devices do not maximize the removal of proteins,lipids, and other cellular components that may inhibit PCR in amicrofluidic system.

Although the various methods exist to capture nuclei for use in downstream application or to separate specific cells from a samplepopulation, none of these methods describes a device that is capable ofextracting cell nuclei and performing PCR assays on the nuclei using thesame device. Thus, there is a need to develop improved systems andmethods for DNA purification through nuclei isolation and integrated PCRdetection of genetic sequences in microfluidic devices.

SUMMARY OF THE INVENTION

The present invention relates to methods and systems for the isolationof DNA on a microfluidic device and the subsequent analysis of the DNAon the microfluidic device. More specifically, embodiments of thepresent invention relate to methods and systems for the isolation of DNAfrom patient samples on a microfluidic device and the use of the DNA forperforming amplification reactions, such as PCR, and detection, such asthermal melt analysis, on the microfluidic device.

In one aspect, the present invention provides a method of isolating DNAfrom cells in a sample. According to this aspect, the method comprises:(a) selectively lysing the cellular membranes of the cells in the samplewithout lysing the nuclear membranes of the cells to produce intactnuclei from the cells; (b) separating the intact nuclei from the sampleby a nuclei size exclusion barrier in a nuclei separation region of amicrofluidic device; (c) resuspending the separated nuclei in an elutionbuffer in the nuclei separation region of the microfluidic device; (d)delivering the resuspended nuclei to a nuclei lysis region of amicrofluidic device and (e) lysing the resuspended nuclei to release theDNA in the nuclei lysis region of the microfluidic device.

In some embodiments, the sample is a patient sample which can be, forexample, a blood sample, a urine sample, a saliva sample, a sputumsample, a cerebrospinal fluid sample, a body fluid sample or a tissuesample. In other embodiments, the patient sample contains white bloodcells. In additional embodiments, the patient sample is a blood samplethat is first enriched for white blood cells prior to the selectivelysis of the cellular membrane. In some embodiments, the enrichment ofwhite blood cells is performed by filtration. In additional embodiments,the enrichment of white blood cells is performed using antibodies. Insome embodiments, the antibodies are coupled to a solid phase, such asbeads, magnetic beads, particles, polymeric beads, chromotagraphicresin, filter paper, a membrane or a hydrogel.

In some embodiments, the selective lysis is performed by contacting thepatient sample, such as whole blood or a sample after white blood cellenrichment, with a nuclei isolation buffer that selectivelypermeabilizes cellular membranes while leaving the nuclei of the cellsintact. In other embodiments, the selective lysis is performed using ahypotonic lysis buffer that contains a weak detergent. In furtherembodiments, the patient sample and the hypotonic lysis buffer are mixedin a 1:1 ratio. In additional embodiments, the selective lysis of thecellular membranes totally lyses red blood cells. In some embodiments,the patient sample and the nuclei isolation buffer are mixed off themicrofluidic device and then added to the nuclei separation region ofthe microfluidic device. In other embodiments, the patient sample andthe nuclei isolation buffer are mixed in a cell lysis region of amicrofluidic device.

In some embodiments, the nuclei separation region in the microfluidicdevice has a nuclei size exclusion barrier for separating the nucleifrom the rest of the patient sample, including the cell debris. In otherembodiments, the nuclei size exclusion barrier is a system of pillarsprefabricated into the nuclei separation region to retard the nuclei onone side of the nuclei separation region. In other embodiments, thenuclei size exclusion barrier is a filter system constructed to retardthe nuclei on one side of the nuclei separation region. In additionalembodiments, the nuclei size exclusion barrier comprises holes along thebottom of the nuclei separation region designed to retard the nuclei onone side of the nuclei separation region. In some embodiments, flowthrough the nuclei size exclusion barrier is driven by a pressuredifferential.

In some embodiments, the elution buffer is a buffer in which the nucleiare compatible. In other embodiments, the elution buffer is anamplification reaction buffer that may contain the non-assay specificamplification reagents. In additional embodiments, the amplificationreaction buffer is a PCR buffer that may contain the non-assay specificPCR reagents. In further embodiments, the elution buffer contains a dyethat binds to DNA. In additional embodiments, the dye is useful forquantifying the amount of DNA in the channel. In some embodiments, theintact nuclei are resuspended in the elution buffer by flow of theelution buffer through the nuclei separation region and across thenuclei size exclusion barrier, which flow is driven by a pressuredifferential. In other embodiments, the resuspended nuclei are driven toa nuclei lysis region of a microfluidic device. In additionalembodiments, the nuclei are lysed by heat in the nuclei lysis region torelease the DNA from the nuclei. In some embodiments, the nuclei aresubjected to heat in a nuclei lysis region prior to an amplificationreaction. In other embodiments, the nuclei are subjected to heat duringthe amplification reaction and the nuclei lysis region is the initialregion of microfluidic device in which the amplification reaction isconducted.

In another aspect, the present invention provides a method ofdetermining presence or absence of a nucleic acid in a patient sample.According to this aspect, the method comprises: (a) selectively lysingthe cellular membranes of cells in the patient sample without lysing thenuclear membranes of the cells to produce intact nuclei from the cells;(b) separating the intact nuclei from the patient sample by a nucleisize exclusion barrier in a nuclei separation region of a microfluidicdevice; (c) resuspending the separated nuclei in an elution buffer inthe nuclei separation region of the microfluidic device; (d) deliveringthe resuspended nuclei to a nuclei lysis region of a microfluidicdevice; (e) lysing the separated nuclei to release the nucleic acid inthe microfluidic device; (f) amplifying the nucleic acid in themicrofluidic device; and (g) determining the presence or absence of anamplified product, wherein the presence of the amplified productindicates the presence of the nucleic acid in the patient sample.

In some embodiments, the patient sample is as described above. In otherembodiments, the patient sample is first enriched for white blood cellsprior to the selective lysis of the cellular membrane as describedabove. In other embodiments, the selective lysis is performed asdescribed above. In additional embodiments, the nuclei separation regionof the microfluidic device has a nuclei size exclusion barrier forseparating the nuclei from the rest of the patient sample as describedabove. In further embodiments, the elution buffer is a buffer asdescribed above. In other embodiments the nuclei are lysed by heat torelease the nucleic acid from the nuclei as described above.

In some embodiments, the resuspended nuclei or the isolated nucleic acidfrom a nuclei lysis region of the microfluidic device is introduced intoa single reaction channel in a microfluidic device for amplification andanalysis. In other embodiments, the resuspended nuclei or the isolatednucleic acid from a nuclei lysis region of the microfluidic device isintroduced into two or more reaction channels in a microfluidic devicefor amplification and analysis. In further embodiments, the resuspendednuclei or the isolated nucleic acid is introduced into the reactionchannels by application of a pressure differential. In some embodiments,amplification reaction buffer that may contain the non-assay specificamplification reagents is added to the resuspended nuclei or theisolated nucleic acid that are in a non-amplification elution bufferprior to introduction into the reaction channels. In additionalembodiments, the quantity of resuspended nuclei or the isolated DNA isdetermined prior to introduction into the reaction channels. In furtherembodiments, assay specific reagents are added to the resuspended nucleior the isolated nucleic acid. In some embodiments, the assay specificreagents are added prior to introduction of the resuspended nuclei orisolated nucleic acid into the reaction channels. In other embodiments,the assay specific reagents are added after introduction of theresuspended nuclei or the isolated nucleic acid into the reactionchannels. In additional embodiments, the amplification reaction is apolymerase chain reaction. In further embodiments, the presence orabsence of amplified product is detected. In some embodiments, thedetection of amplified product is performed by thermal melt analysis. Inother embodiments, the detection of amplified product is performed byusing a label that changes intensity upon the presence of amplifiedproduct.

In some embodiments, steps (b)-(g) and optionally step (a) are performedin one microfluidic device. In other embodiments steps (b) and (c) andoptionally step (a) are performed in one microfluidic device and steps(e)-(g) are performed in a second microfluidic device. In furtherembodiments, steps (b)-(e) and optionally step (a) are performed in onemicrofluidic device and steps (f) and (g) are performed in a secondmicrofluidic device.

In an additional aspect, the present invention provides a microfluidicsystem for isolating DNA from cells in a patient sample. According tothis aspect, the microfluidic system comprises a cell lysis region inwhich the cellular membranes of the cells in the patient sample areselectively lysed without lysing the nuclear membranes of the cells toproduce intact nuclei from the cells. The microfluidic system alsocomprises a nuclei separation region in a microfluidic device whichblocks intact nucleic while passing the rest of the patient sampledriven by a pressure differential to carry away components of thepatient sample smaller than the intact nuclei and the intact nucleiresuspended in an elution buffer being driven by a pressure differentialto carry intact nuclei out of the nuclei size exclusion region. Themicrofluidic system further comprises a nuclei lysis region in which thenuclear membranes of the intact nuclei are lysed to release the DNA.

In some embodiments, the nuclei separation region includes a nuclei sizeexclusion barrier as described above. In other embodiments, the celllysis region is off the microfluidic device. In additional embodiments,the cell lysis region is in a channel in the microfluidic device. Infurther embodiments, the nuclei lysis region comprises a source of heatsufficient to lysis the nuclear membranes of the intact nuclei andrelease the DNA.

In some embodiments, the microfluidic system further comprises a controlsystem which controls the flow of the patient sample through the nucleiseparation region and controls the flow of the elution buffer throughthe nuclei separation region. In other embodiments, the control systemcontrols the flow of the patient sample and elution buffer by vacuumpressure. In further embodiments, the control system causes the patientsample to flow in a first direction and causes the elution buffer toflow in a second direction. In some embodiments, the first direction issubstantially orthogonal to the second direction.

In a further aspect, the present invention provides a microfluidicsystem for determining presence or absence of a nucleic acid in apatient sample. According to this aspect, the microfluidic systemcomprises a cell lysis region, a nuclei separation region and a nucleilysis region as described above. The microfluidic system also comprisesan amplification reaction region in which the nucleic acid is amplified.The microfluidic system further comprises a detection region fordetermining the presence or absence of an amplified product. Themicrofluidic system may further comprise a control system as describedabove.

In some embodiments, the nuclei separation region includes a nuclei sizeexclusion barrier as described above. In other embodiments, the celllysis region is off the microfluidic device. In additional embodiments,the cell lysis region is in a channel in a microfluidic device. Infurther embodiments, the nuclei lysis region comprises a source of heatsufficient to lysis the nuclear membranes of the intact nuclei andrelease the nucleic acid. In other embodiments, the source of heat is inthe amplification reaction region. In some embodiments, theamplification reaction region is a PCR region. In other embodiments, thedetection region is a thermal melt analysis region. In additionalembodiments, the microfluidic system further comprises a nucleic acidquantification region before the amplification reaction region forquantifying the nucleic acid in the channel prior to the amplificationreaction region. In some embodiments, the amplification reaction regionand the detection region comprises multiple channels. In otherembodiments, each channel in the amplification reaction region and thedetection region receives nucleic acid from a single channel containingthe nuclei size exclusion region and the nuclei lysis region. In furtherembodiments, two or more channels in the amplification reaction regionand the detection region receive nucleic acid from a single channelcontaining the nuclei size exclusion region and the nuclei lysis region.

In some embodiments, optionally the cell lysis region and all of theremaining regions are in a single microfluidic device. In otherembodiments, the nuclei separation region and the nuclei lysis regionand optionally the cell lysis region are in one microfluidic device andthe amplification reaction region and the detection region are in asecond microfluidic device. In further embodiments, the nucleiseparation region and optionally the cell lysis region are in onemicrofluidic device and the cell lysis region, amplification reactionregion and detection region are in a second microfluidic device.

The above and other embodiments of the present invention are describedbelow with reference to the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated herein and form partof the specification, illustrate various embodiments of the presentinvention. In the drawings, like reference numbers indicate identical orfunctionally similar elements.

FIG. 1 illustrates a flow chart of a process for sample preparation on amicrofluidic device in accordance with embodiments of the presentinvention.

FIG. 2 illustrates a system for sample preparation on a microfluidicdevice in accordance with embodiments of the present invention.

FIG. 3 illustrates a system for sample preparation on a microfluidicdevice in accordance with other embodiments of the present invention.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The present invention has several embodiments and relies on patents,patent applications and other references for details known to those ofthe art. Therefore, when a patent, patent application, or otherreference is cited or repeated herein, it should be understood that itis incorporated by reference in its entirety for all purposes as well asfor the proposition that is recited.

The practice of the present invention may employ, unless otherwiseindicated, conventional techniques and descriptions of organicchemistry, polymer technology, molecular biology (including recombinanttechniques), cell biology, biochemistry, and immunology, which arewithin the skill of the art. Such conventional techniques includepolymer array synthesis, hybridization, ligation, and detection ofhybridization using a label. Specific illustrations of suitabletechniques can be had by reference to the example herein below. However,other equivalent conventional procedures can, of course, also be used.Such conventional techniques and descriptions can be found in standardlaboratory manuals such as Genome Analysis: A Laboratory Manual Series(Vols. I-IV), Using Antibodies: A Laboratory Manual, Cells: A LaboratoryManual, PCR Primer: A Laboratory Manual, and Molecular Cloning: ALaboratory Manual (all from Cold Spring Harbor Laboratory Press),Stryer, L. (1995) Biochemistry (4th Ed.) Freeman, N.Y., Gait,Oligonucleotide Synthesis: A Practical Approach, 1984, IRL Press,London, Nelson and Cox (2000), Lehninger, Principles of Biochemistry 3rdEd., W. H. Freeman Pub., New York, N.Y. and Berg et al. (2002)Biochemistry, 5th Ed., W. H. Freeman Pub., New York, N.Y., all of whichare herein incorporated in their entirety by reference for all purposes.

The present invention provides methods and systems for the isolation ofDNA from patient samples on a microfluidic device and the subsequentanalysis of the DNA on a microfluidic device. More specifically, thepresent invention provides methods and systems for the isolation of DNAfrom patient samples on a microfluidic device and the use of the DNA forperforming amplification reactions, such as PCR, and detection, such asthermal melt analysis, on a microfluidic device.

Thus, in a first aspect, the present invention provides a method ofisolating DNA from cells in a patient sample comprising: (a) selectivelylysing the cellular membranes of the cells in the patient sample withoutlysing the nuclear membranes of the cells to produce intact nuclei fromthe cells; (b) separating the intact nuclei from the patient sample by anuclei size exclusion barrier in a nuclei separation region of amicrofluidic device; (c) resuspending the separated nuclei in an elutionbuffer in the nuclei separation region of the microfluidic device; (d)delivering the resuspended nuclei to a nuclei lysis region of amicrofluidic device; and (e) lysing the resuspended nuclei to releasethe DNA in the nuclei lysis region of the microfluidic device.

The selective lysis involves lysing the cellular membranes of the cellswithout lysing the nuclear membranes of the cells in the patient sampleto produce intact nuclei from the cells. The patient sample may be ablood sample, a urine sample, a saliva sample, a sputum sample, acerebrospinal fluid sample, a body fluid sample or a tissue sample. Insome embodiments, the patient sample contains white blood cells. In apreferred embodiment, the patient sample is a blood sample. Since redblood cells only have a cellular membrane and do not include a nucleus,the selective lysis totally lyses the red blood cells in a patient bloodsample. In some embodiments, the patient sample is first enriched forwhite blood cells prior to the selective lysis of the cellularmembranes. Techniques for the enrichment of white blood cells are knownto the skilled artisan. In some embodiments, the enrichment of whiteblood cells is performed by filtration. In additional embodiments, theenrichment of white blood cells is performed using antibodies. In someembodiments, the antibodies art coupled to a solid phase, such as beads,magnetic beads, particles, polymeric beads, chromotagraphic resin,filter paper, a membrane or a hydrogel. See, for example, U.S. Pat. Nos.4,752, 564, 5,118,428, 5,482,829 and 5,736,033, PCT InternationalPublication No. WO 88/05331, Boyum (Nature 204:793-794, 1964) andVanDelinder and Groisman (Anal Chem 79:2023-2030, 2007). Products thatare commercially available for the enrichment of white blood cellsinclude, but are not limited to, Ficoll-Paque™ PLUS (GE Healthcare,Piscataway, N.J.), White Blood Cell Isolation (Leukasorb) Medium (PallCorporation, East Hills, N.Y.) and Accuspin™ System (Sigma Aldrich, St.Louis, Mo.).

The selective lysis is performed by contacting the patient sample with abuffer (referred to herein as a selective lysis buffer, a lysis bufferor a nuclei isolation buffer) that selectively permeabilizes cellularmembranes while leaving the nuclei of the cells intact. Nuclei isolationbuffers that have these characteristics are well known to the skilledartisan. Products that include nuclei isolation buffers for selectivelylysing cellular membranes are commercially available. Suitablecommercial products that include such buffers, include, but are notlimited to, Nuclei EZ Prep Nuclei Isolation Kit (NUC-101) (Sigma, St.Louis, Mo., USA), Nuclear/Cytosol Fractionation Kit (K266-100)(BioVision Research Products, Mountain View, Calif., USA), NE-PERNuclear and Cytoplasmic Extraction Reagents (Pierce, Rockville, Ill.,USA), Nuclear Extraction Kit (Imgenex, Corp., San Diego, Calif., USA),Nuclear Extract Kit (Active Motif, Carlsbad, Calif., USA), and QproteomeNuclear Protein Kit (Qiagen, Valencia, Calif., USA). See also, U.S. Pat.Nos. 5,447,864, 6,852,851 and 7,262,283. It is known that the type ofnuclei in question may determine which nuclei isolation buffer will berequired. See, U.S. Pat. No. 5,447,864 for a discussion of factors thatcan be optimized for preparing a suitable selective lysis buffer fordifferent cell types.

In one embodiment, the selective lysis buffer is a hypotonic buffer. Forexample, commercial hypotonic lysis buffer can be purchased from SigmaAldrich, Nuclei EZ lysis buffer (N 3408). A kit is also available fromSigma Aldrich, Nuclei EZ Prep Nuclei Isolation Kit (Nuc-101). A commonrecipe for a 10× hypotonic solution is, 100 mM HEPES, pH 7.9, with 15 mMMgCl₂ and 100 mM KCl. In another embodiment, the buffer is a hypotonicbuffer that comprises a detergent. Suitable detergents include, but arenot limited to ionic detergents, such as lithium lauryl sulfate, sodiumdeoxycholate, and Chaps, or non-ionic detergents, such as Triton X-100,Tween 20, Np-40, and IGEPAL CA-630. In another embodiment, the buffer isan isotonic buffer. For example, Sigma Aldrich offers a kit, CelLyticNuclear Extraction kit, which contains an isotonic lysis buffer. Acommon recipie for a 5× isotonic lysis buffer is, 50 mM Tris HCl, pH7.5, with 10 mM MgCl₂, 15 mM CaCl₂, and 1.5M Sucrose. In an additionalembodiment, the buffer is an isotonic buffer that comprises a detergentwhich may be an ionic detergent or a non-ionic detergent.

The patient sample and lysis buffer are contacted for a period of timesufficient to effect the selective lysis. A suitable period of time mayrange from 1-10 minutes, preferably 2-8 minutes, more preferably 3-5minutes. If the patient sample is a blood sample, the period of time issufficient to totally lyse the red blood cells. In some embodiments, thepatient sample and lysis buffer may be mixed in accordance with themanufacturers' recommendations. In some embodiments, the patient sampleand lysis buffer are mixed in a 1:1 ratio. In other embodiments, thepatient sample and lysis buffer are contacted at room temperature. Insome embodiments, the patient sample and lysis buffer are contacted,such as by mixing, off a microfluidic device. This contacting couldoccur in vial, tube, microtiter plate, and the like.

In some embodiments, the patient sample and lysis buffer are contactedin a microfluidic device. In one embodiment, the patient sample andlysis buffer are added to a cell lysis region of the microfluidicdevice. This cell lysis region may be a chamber or a channel. In anotherembodiment, the patient sample can be added to one port of amicrofluidic device and the lysis buffer added to a second port. Thepatient sample and lysis buffer are then flowed together in a cell lysisregion of the microfluidic device. The flow of the patient sample, lysisbuffer and the contacted mixture can be driven by a pressuredifferential. Examples of techniques for contacting the patient sampleand lysis buffer in a microfluidic device are disclosed in U.S.application Ser. No. 12/505,202, filed on Jul. 17, 2009, entitled“METHODS AND SYSTEMS FOR MICROFLUIDIC DNA SAMPLE PREPARATION,” namingWeidong Cao, Hiroshi Inoue and Kevin Louder as inventors, incorporatedherein by reference.

In those embodiments in which the patient sample and lysis buffer arecontacted off a microfluidic device, the selectively lysed sample isadded to a nuclei separation region of a microfluidic device. In thoseembodiments in which the patient sample and the lysis buffer arecontacted in a microfluidic device, the selectively lysed sample isdriven to a nuclei separation region of the microfluidic device. Thenuclei separation region may be a chamber or a channel in a microfluidicdevice. The nuclei separation region includes a nuclei size exclusionbarrier that is capable of separating the intact nuclei from theremainder of the rest of the patient sample, including cell debris. Insome embodiments, the nuclei size exclusion barrier is a system ofpillars prefabricated into the nuclei separation region to retard thenuclei on one side of the nuclei separation region. In otherembodiments, the nuclei size exclusion barrier is a filter systemconstructed to retard the nuclei on one side of the nuclei separationregion. In additional embodiments, the nuclei size exclusion barriercomprises holes along the bottom of the nuclei separation regiondesigned to retard the nuclei on one side of the nuclei separationregion. In some embodiments, flow through the nuclei size exclusionbarrier is driven by a pressure differential.

In some embodiments, the nuclei exclusion barrier has an exclusion sizein the range of 2.7 μm to 5.0 μm. In other embodiments, the nucleiexclusion barrier has an exclusion size in the range of 2.7 μm to 4 μm.In additional embodiments, the nuclei exclusion barrier has an exclusionsize in the range of 2.7 μm to 3 μm. In further embodiments, the nucleiexclusion barrier has an exclusion size of 2.7 μm.

After the intact nuclei have been separated from the rest of the patientsample including the cell debris, the intact nuclei are resuspended inan elution buffer. The elution buffer is one in which the nuclei arecompatible and one that is compatible with any downstream processing ofthe DNA that is to be isolated from the nuclei. If the DNA is to besubjected to an amplification reaction, the elution buffer may be anamplification reaction buffer. In some embodiments, the amplificationreaction buffer may contain the non-assay specific amplificationreagents. If the amplification reaction is a polymerase chain reaction,the buffer may be a PCR buffer that may contain the non-specific PCRreagents. In some embodiments, the elution buffer also contains a dyethat binds to DNA. The DNA dye may be one that binds to free DNA or itmay be one that binds to chromosomal DNA, that is, the dye is able tocross the nuclear membrane and bind to the DNA in the intact nuclei. DNAdyes are well known to the skilled artisan. Examples of DNA dye include,but are not limited to, 4′,6-diamidino-2-phenylindole (DAPI), SYBR®Green, SYBR® GreenER™ and PicoGreen (Invitrogen Corp., Carlsbad, Calif.)and LC Green (Idaho Technology, Salt Lake City, Utah). In someembodiments, the DNA dye is useful for quantifying the amount of DNAthat has been isolated or that is to be used for downstream processing.

In some embodiments, the intact nuclei are resuspended in the elutionbuffer by flow of the elution buffer through the nuclei separationregion and across the nuclei size exclusion barrier, which flow isdriven by a pressure differential. Once the intact nuclei have beenresuspended in the elution buffer, the resuspended nuclei are driven toa nuclei lysis region of a microfluidic device. In one embodiment, thenuclei are lysed by heat in the nuclei lysis region to release the DNAfrom the nuclei. In some embodiments, the nuclei are subjected to heatin a nuclei lysis region prior to an amplification reaction. In otherembodiments, the nuclei are subjected to heat during the amplificationreaction, and the nuclei lysis region is in the initial portion of amicrofluidic device in which the amplification reaction is conducted. Insome embodiments, the nuclei lysis region is in the same microfluidicdevice as the nuclei separation region. In other embodiments, the nucleilysis region is in a different microfluidic chip than the nucleiseparation region.

In a second aspect, the present invention provides a method ofdetermining the presence or absence of a nucleic acid in a patientsample comprising: (a) selectively lysing the cellular membranes of thecells in the patient sample without lysing the nuclear membranes of thecells to produce intact nuclei from the cells; (b) separating the intactnuclei from the patient sample by a nuclei size exclusion barrier in anuclei separation region of a microfluidic device; (c) resuspending theseparated nuclei in an elution buffer in the nuclei separation region ofthe microfluidic device; (d) delivering the resuspended nuclei to anuclei lysis region of a microfluidic device; (e) lysing the resuspendednuclei to release the nucleic acid in the nuclei lysis region of themicrofluidic device; (f) amplifying the nucleic acid in a microfluidicdevice; and (g) determining the presence or absence of an amplifiedproduct.

In some embodiments, the patient sample is as described above. In otherembodiments, the patient sample is first enriched for white blood cellsprior to the selective lysis of the cellular membrane as describedabove. In some embodiments, the selective lysis is performed asdescribed above. In additional embodiments, the nuclei separation regionof the microfluidic device has a nuclei size exclusion barrier forseparating the nuclei from the rest of the patient sample as describedabove. In further embodiments, the elution buffer is a buffer asdescribed above. In other embodiments the nuclei are lysed by heat torelease the nucleic acid from the nuclei as described above.

After lysing the nuclei to release the nucleic acid, the nucleic acid isthen subjected to an amplification reaction. As described above, thislysis may be performed in the initial phase of the amplificationreaction. In some embodiments, the amplification reaction is apolymerase chain reaction. In other embodiments, the amplificationreaction is a real-time polymerase chain reaction. These polymerasechain reactions, as well as other amplification reactions, are wellknown to the skilled artisan.

In some embodiments, the resuspended nuclei or the isolated nucleic acidfrom a nuclei lysis region of the microfluidic device is introduced intoa single reaction channel in a microfluidic device for amplification andanalysis. In other embodiments, the resuspended nuclei or the isolatednucleic acid from a nuclei lysis region of the microfluidic device isintroduced into two or more reaction channels in a microfluidic devicefor amplification and analysis. In further embodiments, the resuspendednuclei or isolated nucleic acid is introduced into the reaction channelsby application of a pressure differential. In some embodiments,amplification reaction buffer that may contain the non-assay specificamplification reagents is added to the resuspended nuclei or theisolated nucleic acid in a non-amplification elution buffer prior tointroduction into the reaction channels. In additional embodiments, thequantity of resuspended nuclei or the isolated nucleic acid, such asDNA, is determined prior to introduction into the reaction channels. Infurther embodiments, assay specific reagents are added to theresuspended nuclei or the isolated nucleic acid. In some embodiments,the assay specific reagents are added prior to introduction of theresuspended nuclei or the isolated nucleic acid into the reactionchannels. In other embodiments, the assay specific reagents are addedafter introduction of the resuspended nuclei or the isolated nucleicacid into the reaction channels. In some embodiments, the pressuredifferential, such as a vacuum, is applied from the end of the reactionchannels and pulls the elution buffer, which may be PCR reaction buffer,into the nuclei separation chamber to resuspend the nuclei. In otherembodiments, the pressure differential also pulls the PCR reactionbuffer, if it is not the elution buffer, and the assay specific reagentsto mix with the resuspended nuclei before the reaction mixture is pulledinto the reaction channels. In additional embodiments, the amplificationreaction is a polymerase chain reaction.

In some embodiments, the presence or absence of amplified product isdetected. In other embodiments, the detection of amplified product isperformed by thermal melt analysis. In further embodiments, thedetection of amplified product is performed by using a label thatchanges intensity upon the presence of amplified product. Methods forthe detection of amplified products, including thermal melt analysis,are well known to the skilled artisan.

In some embodiments, steps (b)-(g) and optionally step (a) are performedin one microfluidic device. In other embodiments steps (b) and (c) andoptionally step (a) are performed in one microfluidic device and steps(e)-(g) are performed in a second microfluidic device. In furtherembodiments, steps (b)-(e) and optionally step (a) are performed in onemicrofluidic device and steps (f) and (g) are performed in a secondmicrofluidic device. Microfluidic methods and systems for amplificationand detection of nucleic acids are known in the art. See, for example,U.S. Patent Application Publication Nos. 2005/0042639, 2008/0003588,2008/0003593, 2008/0176230, 2009/0053726 and 2009/0111149, eachincorporated herein by reference.

FIG. 1 is a flow chart illustrating a process 100 for the isolation ofDNA from a patient sample (such as blood sample), amplification of theisolated DNA and analysis of amplified products in accordance with oneembodiment of the present invention. In step 101, whole blood samplefrom a patient is obtained. In step 102, a hypotonic lysis buffer isadded to the whole blood sample, for example, in a ratio of 1:1 (v:v),and incubated at room temperature for approximately three minutes toobtain a lysed whole blood sample with intact nucleic. In step 103, theintact nuclei are separated from the lysed whole blood sample. In oneembodiment, a predetermined amount of the lysed blood sample with intactnuclei are delivered to a nuclei separation region of a microfluidicdevice and a pressure differential (such as a vacuum) is applied in adirection that will allow for the removal of all of the samplecomponents except intact nuclei through a nuclei size exclusion barrierinto a waste reservoir.

In step 104, the intact nuclei are resuspended in an elution buffer. Inone embodiment, a pressure differential is applied in an alternatedirection to that in step 103 to pull the elution buffer from areservoir into the nuclei separation region to resuspend the intactnuclei. In one embodiment, the elution buffer can be a PCR reactionbuffer. In step 105, the resuspended intact nuclei are delivered to anucleic lysis region of a microfluidic device and the nuclei are lysedto release the DNA. In one embodiment, the pressure differential used instep 103 delivers the resuspended nuclei to the nuclei lysis region. Inanother embodiment, PCR assay specific reagents are pulled from areagent reservoir into the resuspended nuclei/elution buffer mixture bythe same pressure differential (such as a vacuum). The released DNA isthen introduced into an amplification region of microfluidic device andan amplification reaction, such as a polymerase chain reaction, isperformed in step 106. In step 107, the PCR products are analyzed, suchas by a thermal melt analysis.

In a third aspect, the present invention provides a microfluidic systemfor isolating DNA from cells in a patient sample. According to thisaspect, the microfluidic system comprises a cell lysis region in whichthe cellular membranes of the cells in the patient sample areselectively lysed without lysing the nuclear membranes of the cells toproduce intact nuclei from the cells. The microfluidic system alsocomprises a nuclei separation region in a microfluidic device whichblocks the passage of intact nucleic while passing the rest of thepatient sample driven by a pressure differential to carry awaycomponents of the patient sample smaller than the intact nuclei, andwherein the intact nuclei resuspended in an elution buffer are driven bya pressure differential to carry intact nuclei out of the nuclei sizeexclusion region. The microfluidic system further comprises a nucleilysis region in which the nuclear membranes of the intact nuclei arelysed to release the DNA.

An example of a suitable system for use in accordance with certainaspects of the present invention is illustrated in connection with FIG.2. As illustrated in FIG. 2, the system includes a cell lysis regionwhich includes a sample chamber 201, a lysis buffer chamber 202 and acell lysis chamber 203. As illustrated in FIG. 2, the cell lysis regionmay be off a microfluidic device. It is also contemplated that the celllysis region may be on a microfluidic device. In this embodiment, thesample chamber and lysis buffer chambers are in a microfluidic deviceand flow into a connected channel to mix together in a cell lysisportion of the channel. Examples of cell lysis regions in a microfluidicdevice are disclosed in U.S. application Ser. No. 12/505,202, filed onJul.17, 2009, entitled “METHODS AND SYSTEMS FOR MICROFLUIDIC DNA SAMPLEPREPARATION,” naming Weidong Cao, Hiroshi Inoue and Kevin Louder asinventors, incorporated herein by reference.

Returning to the system shown in FIG. 2, after lysis, the sample isdelivered through port 204 to a nuclei separation region 205 of amicrofluidic device 200. If the cell lysis region is in the microfluidicdevice, the sample is delivered to the nuclei separation region. Thenuclei separation region 205 includes a nuclei size exclusion barrier207 for separating intact nuclei 206 from the waste which passes towaste reservoir 208. The waste includes all of the components of thesample other than the intact nuclei. The nuclei size exclusion barrieris as described above. As illustrated in FIG. 2, the system alsocomprises an elution buffer port 209 in fluid communication with thenuclei separation region 205 for providing elution buffer to the nucleiseparation region 205. Introduction of the elution buffer by, forexample, a pressure differential resuspends the isolated intact nucleiand carries the resuspended nuclei out of the nuclei separation region205 and into a nuclei lysis region 210.

As illustrated in FIG. 2, the system includes the nuclei lysis region210 in which the nuclear membranes of the intact nuclei are lysed torelease the DNA. The nuclei lysis region 210 is in fluid communicationwith the nuclei separation region 205. The nuclei lysis region mayinclude a heat source, such as a lamp 212, sufficient to lyse thenuclear membranes. The system may further contain an assay specificreagent port 211 in fluid communication with the nuclei lysis region 210for the introduction of amplification assay specific reagents into theelution buffer containing the intact nuclei or the released DNA. Theassay specific reagent port 211 may be located upstream or downstream ofthe heat source.

The system may also include a control system which controls the flow ofthe patient sample through the nuclei separation region, the flow of theelution buffer through the nuclei separation region, the flow of theassay specific reagents to the nuclei lysis region and the heat suppliedto the nuclei lysis region. In some embodiments, the control systemcomprises a main controller 210 which communicates with a flow controlsystem 214 and a temperature control system 216. As those skilled in theart will recognize, many options exist for the main controller 210, oneexample being a general purpose computer and another being a specialpurpose computer. Other specialized control equipment in the prior artcould also serve the purpose of the main controller 210.

In some embodiments, the flow control system 214 controls the flow ofthe patient sample through the port 204 and into the nuclei separationregion 205. Flow control system 214 also controls the flow of the wastematerial through the nuclei size exclusion barrier 207 and into thewaste reservoir 208 such as, for example, by vacuum port 219. Flowcontrol system 214 further controls the flow of the elution bufferthrough the elution buffer port which resuspends the isolated intactnuclei and carries the resuspended nuclei out of the nuclei separationregion 205 and into the nuclei lysis region 210. In other embodiments,flow control system controls 214 controls the flow of the assay specificreagents through port 211 and into the nuclei lysis region 210 for theintroduction of amplification assay specific reagents into the elutionbuffer containing the intact nuclei or the released DNA.

In some embodiments, the flow control system 214 controls the flow ofthe patient sample and elution buffer by a pressure differential, suchas by vacuum pressure. In other embodiments, the flow control system 214causes the patient sample to flow in a first direction and causes theelution buffer to flow in a second direction. In some embodiments, thefirst direction is substantially orthogonal to the second direction. Inother embodiments, a valve 213, shown in FIG. 2, separates the nucleiseparation region 205 and the nuclei lysis region 210 which iscontrolled by the flow control system 214 to permit the resuspendednuclei and elution buffer to pass from the nuclei separation region tothe nuclei lysis region 210.

In some embodiments, the temperature control system 216 controls thetemperature of heat source 212 sufficient to lyse the nuclear membranesof the intact nuclei in the lysis region 210.

In another aspect, the present invention provides a microfluidic systemfor determining presence or absence of a nucleic acid in a patientsample. According to this aspect, the microfluidic system comprises acell lysis region, a nuclei separation region and a nuclei lysis regionas described above. The microfluidic system may further comprise acontrol system as described above. The microfluidic system alsocomprises an amplification reaction region in which the nucleic acid isamplified. The microfluidic system further comprises a detection regionfor determining the presence or absence of an amplified product.

An example of a suitable system for use in accordance with certainaspects of the present invention is illustrated in connection with FIG.3. As illustrated in FIG. 3, the system includes a cell lysis regionwhich includes a sample chamber 301, a lysis buffer chamber 302 and acell lysis chamber 303. As illustrated in FIG. 3, the cell lysis regionmay be off a microfluidic device. It is also contemplated that the celllysis region may be included on or in a microfluidic device. In oneembodiment, the sample chamber and lysis buffer chambers are in amicrofluidic device and flow into a connected channel to mix together ina cell lysis portion of the channel. Examples of cell lysis regions in amicrofluidic device are disclosed in U.S. application Ser. No.12/505,202, filed on Jul. 17, 2009, entitled “METHODS AND SYSTEMS FORMICROFLUIDIC DNA SAMPLE PREPARATION,” naming Weidong Cao, Hiroshi Inoueand Kevin Louder as inventors, incorporated herein by reference.

Returning to the system shown in FIG. 3, after cell lysis, the sample isdelivered through port 304 to a nuclei separation region 305 of themicrofluidic device. If the cell lysis region is in the microfluidicdevice, the sample is delivered to the nuclei separation region 305. Thenuclei separation region 305 includes a nuclei size exclusion barrier307 for separating intact nuclei 306 containing nucleic acid from thewaste which passes to waste reservoir 308. In preferred embodiments, thewaste includes all of the components of the sample other than the intactnuclei. The nuclei size exclusion barrier is as described above. Asillustrated in FIG. 3, the system also comprises an elution buffer port309 in fluid communication with the nuclei separation region 305 forproviding elution buffer to the nuclei separation region 305. The systemmay contain a second elution buffer port 318 in fluid communication withthe nuclei separation region 305.

In operation, the sample is delivered through port 304 to the nucleiseparation region 305 of the microfluidic device. In one embodiment, avaccum is then applied in the Y-direction, such as, for example, fromthe vacuum port 319 located in the waste reservoir 308. This will pullall the sample waste into the waste reservoir while the intact nucleiare prevented from passing to the waste reservoir by the nuclei sizeexclusion barrier 307. A vacuum is then applied in the X-direction, suchas, for example, by the vacuum port 325 located at the end of themicrofluidic channels 330a. This will allow for the introduction of theelution buffer from the elution buffer port 309 into the nucleiseparation region 305 to resuspend the isolated intact nuclei and tocarry the resuspended nuclei out of the nuclei separation region 305, asdescribed above in connection with FIG. 2.

In another embodiment, the nuclei separation region 305 is filled withelution buffer through either elution buffer port 309 or 318 prior tothe delivery of the lysed sample to the nuclei separation region. In oneembodiment, the nuclei separation region 305 is pre-filled with elutionbuffer by applying pressure in the X-direction to reduce the productionof bubbles. The lysed sample mixture is then delivered to the nucleiseparation region 305 in the Y-direction, either by pressure or vacuum,to allow the separation of the nuclei from the waste and removal of thewaste from the nuclei separation region 305. In one embodiment, theelution buffer port 318 is included to ensure that no bubbles arecreated after delivery of the lysed sample to the nuclei separationregion 305. As shown in FIG. 3, vacuum can be applied in the Y-directionusing vacuum port 319. Introduction of the elution buffer by, forexample, a pressure differential, from elution buffer port 309 afterseparation of the nuclei resuspends the isolated intact nuclei andcarries the resuspended nuclei out of the nuclei separation region 305,as described above in connection with FIG. 2.

Returning to FIG. 3, the system also includes a nuclei lysis region 312in which the nuclear membranes of the intact nuclei are lysed to releasethe DNA. The nuclei lysis region 312 is in fluid communication with thenuclei separation region 305 through, in one embodiment, a valve 315which is controlled by a flow control system (not shown). The nucleilysis region may include a heat source 314, such as a lamp, sufficientto lyse the nuclear membranes. The system may further contain an assayspecific reagent port or sipper 313 in fluid communication with thenuclei lysis region 312 for the introduction of amplification assayspecific reagents 311, such as, for example, assay specific primers andprobes, into the elution buffer containing the intact nuclei or thereleased DNA. The assay specific reagent port or sipper 313 may belocated upstream or downstream of the heat source 314. In oneembodiment, heat from the heat source 314 degrades the nuclei whichallows for DNA to be mixed with PCR buffer and assay specific reagentsprior to loading into the channels 330 a.

As illustrated in FIG. 3, the microfluidic system may further comprise anucleic acid quantification system for quantifying the nucleic acid inthe channel prior to the amplification reaction region. In oneembodiment, a detector 320 is aligned with the nuclei lysis region 312to count the number of nuclei that are directed to one or more channels330 a, in order to know the exact concentration of template beingincluded in the amplification reaction. Although FIG. 3 shows thenucleic acid quantification system aligned with the nuclei lysis region312, it is contemplated that the nucleic acid quantification systemcould also be upstream or downstream of the nuclei lysis region 312. Inone embodiment, the nucleic acid is quantified using a dye that binds tofree DNA or chromosomal DNA, that is, the dye is able to cross thenuclear membrane and bind to the DNA in the intact nuclei. If thenucleic acid is to be quantified, the dye is added to the elutionbuffer. DNA dyes are well known to the skilled artisan. Examples of DNAdye include, but are not limited to, 4′,6-diamidino-2-phenylindole(DAPI), SYBR® Green, SYBR® GreenER™ and PicoGreen (Invitrogen Corp.,Carlsbad, Calif.) and LC Green (Idaho Technology, Salt Lake City, Utah).

As illustrated in FIG. 3, the nucleic acid is delivered into multiplechannels 330a for downstream processing. However, it is contemplatedthat the nucleic acid could be delivered into a single channel fordownstream processing. Also as illustrated in FIG. 3, the nuclei lysisregion is upstream of channels 330a. However, it is contemplated thatthe nuclei lysis region could also be located in the upstream portion ofchannel 330a. In accordance with this aspect of the invention, thedownstream processing may include the amplification of the nucleic acidand the detection of the amplified product. The amplification of thenucleic acid occurs in channel 330 a in an amplification region (notshown) of the microfluidic device. The detection of the amplifiedproduct occurs in channel 330 a in a detection region (not shown) of themicrofluidic device. In some embodiments, the amplification is PCRamplification and the detection is a thermal melt analysis. Microfluidicsystems for amplification and detection of nucleic acids are well knownin the art. See, for example, U.S. Patent Application Publication Nos.2005/0042639, 2008/0003588, 2008/0003593, 2008/0176230, 2009/0053726 and2009/0111149, each incorporated herein by reference.

The system may also include a control system (not shown) which controlsthe flow of the patient sample through the nuclei separation region, theflow of the elution buffer through the nuclei separation region, theflow of the sample into the microchannels, and the temperature of theheat source 314. In some embodiments, the control system controls theflow of the patient sample and elution buffer by vacuum pressure. Inother embodiments, the control system causes the patient sample to flowin a first direction and causes the elution buffer to flow in a seconddirection. In some embodiments, the first direction is substantiallyorthogonal to the second direction. In other embodiments, the controlsystem also controls flow of the sample through the amplification regionand the detection region. A suitable control system for use with theembodiment of FIG. 3 may comprise a main controller, a flow controlsystem and a temperature control system, substantially as describedabove in connection with FIG. 2.

As illustrated in FIG.3, the microfluidic system may include a singledevice with a cell lysis region locate off of the device. However asdescribed above, it is contemplated that the cell lysis region couldalso be in the microfluidic device, such that all of the regions of thesystem are on a single device. It is also contemplated that themicrofluidic system in accordance with certain aspects of the inventionincludes two devices with the cell lysis region off or on a device. Inaccordance with these certain aspects, it is contemplated that thenuclei separation region, the nuclei lysis region and optionally thecell lysis region are on one device and the amplification region and thedetection region are on a second device. It is also contemplated thatthe nuclei separation region, the nuclei lysis region, the nucleic acidquantification region and optionally the cell lysis region are on onedevice and the amplification region and the detection region are on asecond device. It is further contemplated that the nuclei separationregion and optionally the cell lysis region are on one device and thenuclei lysis region, the amplification region and the detection regionare on a second device. In addition, it is contemplated that the nucleiseparation region, the nucleic acid quantification region and optionallythe cell lysis region are on one device and the nuclei lysis region, theamplification region and the detection region are on a second device.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention are to be construed to cover boththe singular and the plural, unless otherwise indicated herein orclearly contradicted by context. The terms “comprising,” “having,”“including,” and “containing” are to be construed as open-ended terms(i.e., meaning “including, but not limited to,”) unless otherwise noted.Recitation of ranges of values herein are merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range, unless otherwise indicated herein, and eachseparate value is incorporated into the specification as if it wereindividually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g., “such as”) provided herein, isintended merely to better illuminate the invention and does not pose alimitation on the scope of the invention unless otherwise claimed. Nolanguage in the specification should be construed as indicating anynon-claimed element as essential to the practice of the invention.

Various embodiments of this invention are described herein. Variationsof those embodiments may become apparent to those of ordinary skill inthe art upon reading the foregoing description. The inventors expectskilled artisans to employ such variations as appropriate, and theinventors intend for the invention to be practiced otherwise than asspecifically described herein. Accordingly, this invention includes allmodifications and equivalents of the subject matter recited in theclaims appended hereto as permitted by applicable law. Moreover, anycombination of the above-described elements in all possible variationsthereof is encompassed by the invention unless otherwise indicatedherein or otherwise clearly contradicted by context.

Additionally, while the processes described above and illustrated in thedrawings are shown as a sequence of steps, this was done solely for thesake of illustration. Accordingly, it is contemplated that some stepsmay be added, some steps may be omitted, the order of the steps may bere-arranged, and some steps may be performed in parallel.

What is claimed is:
 1. A method of isolating DNA from cells in a samplecomprising the steps of: (a) selectively lysing the cellular membranesof the cells in the sample without lysing the nuclear membranes of thecells to produce intact nuclei from the cells; (b) separating the intactnuclei from the sample by a nuclei size exclusion barrier in a nucleiseparation region of a microfluidic device; (c) resuspending theseparated nuclei in an elution buffer in the nuclei separation region ofthe microfluidic device; (d) delivering the resuspended nuclei to anuclei lysis region of a microfluidic device; and (e) lysing theresuspended nuclei in the nuclei lysis region of the microfluidic deviceto release the DNA.
 2. The method of claim 1, wherein the samplecomprises a patient sample comprising whole blood.
 3. The method ofclaim 2, wherein the selective lysis of the cellular membranes totallylyses the red blood cells.
 4. The method of claim 2, wherein the patientsample is first enriched for white blood cells prior to the selectivelysis of the cellular membranes.
 5. The method of claim 1, wherein step(a) is performed off the microfluidic device.
 6. The method of claim 1,wherein step (a) is performed in the microfluidic device.
 7. The methodof claim 1, wherein flow through the nuclei size exclusion barrier isdriven by a pressure differential.
 8. The method of claim 1, wherein theelution buffer is an amplification reaction buffer.
 9. The method ofclaim 1, wherein the nuclei are lysed by heat.
 10. A method ofdetermining the presence or absence of a nucleic acid in a patientsample comprising the steps of: (a) selectively lysing the cellularmembranes of cells in the patient sample without lysing the nuclearmembranes of the cells to produce intact nuclei from the cells; (b)separating the intact nuclei from the patient sample by a nuclei sizeexclusion barrier in a nuclei separation region of a microfluidicdevice; (c) resuspending the separated nuclei in an elution buffer inthe nuclei separation region of the microfluidic device; (d) deliveringthe resuspended nuclei to a nuclei lysis region of a microfluidicdevice; (e) lysing the resuspended nuclei in the nuclei lysis region ofthe microfluidic device to release the nucleic acid; (f) amplifying thenucleic acid in a microfluidic device; and (g) determining the presenceor absence of an amplified product, wherein the presence of theamplified product indicates the presence of the nucleic acid in thepatient sample.
 11. The method of claim 10, wherein the patient sampleis whole blood.
 12. The method of claim 11, wherein the patient sampleis first enriched for white blood cells prior to the selective lysis ofthe cellular membranes.
 13. The method of claim 10, wherein step (a) isperformed off the microfluidic device.
 14. The method of claim 10,wherein step (a) is performed in the microfluidic device.
 15. The methodof claim 10, wherein flow through the nuclei size exclusion barrierdriven by a pressure differential.
 16. The method of claim 10, whereinthe elution buffer is an amplification reaction buffer.
 17. The methodof claim 10, wherein the nuclei are lysed by heat.
 18. The method ofclaim 14, wherein the heat is applied as part of the amplificationreaction.
 19. The method of claim 10, wherein steps (b) and (c) areperformed in one microfluidic device and steps (f) and (g) are performedin a second microfluidic device.